Pancreatic ductal adenocarcinoma (PDAC) suffers from lack of an effective diagnostic method, which hampers improvement in patient survival. Carbohydrate antigen 19-9 (CA19-9) is the only FDA-approved blood biomarker for PDAC, yet its clinical utility is limited due to suboptimal performance. Liquid chromatography-mass spectrometry (LC-MS) has emerged as a burgeoning technology in clinical proteomics for discovery, verification, and validation of novel biomarkers. A plethora of protein biomarker candidates for PDAC have been identified using LC-MS, yet few has successfully transitioned into clinical practice. This translational standstill is owed partly to insufficient considerations of practical needs and perspectives of clinical implementation during biomarker development pipelines, such as demonstrating analytical robustness of proposed biomarkers which is critical for transitioning from research-grade to clinical-grade assays. Moreover, throughput and cost-effectiveness of proposed assays ought to be considered concomitantly from early phases of the biomarker pipelines for enhancing widespread adoption in clinical settings. Here, we developed a fit-for-purpose multi-marker panel for PDAC diagnosis by consolidating analytically robust biomarkers as well as employing a relatively simple LC-MS protocol. In discovery phase, we comprehensively surveyed putative PDAC biomarkers from both in-house data and prior studies. In verification phase, we developed a multiple-reaction monitoring (MRM)-MS-based proteomic assay using surrogate peptides that passed stringent analytical validation tests. We adopted a high-throughput protocol including short gradient (<10 min) and simple sample preparation (no depletion or enrichment steps). Additionally, we developed our assay using serum samples, which are usually the preferred biospecimen in clinical settings. We developed predictive models based on our final panel of 12 protein biomarkers combined with CA19-9, which showed improved diagnostic performance compared to using CA19-9 alone in discriminating PDAC from non-PDAC controls including healthy individuals and patients with benign pancreatic diseases. A large-scale clinical validation is underway to demonstrate the clinical validity of our novel panel.

Endogenous peptides of the 15 biomarker candidates in individual samples of the verification cohort and corresponding spiked stable isotope-labeled (SIL) peptides were analyzed by MRM-MS on a QTRAP 5500+ (Sciex). SIL peptides were obtained from ANYGEN, Korea, and BIOSTEM, Korea. Tryptic peptides were separated on a ZORBAX 300SB-C18 reverse phase column (0.5 × 150 mm, 3.5 μm; Agilent) over 9.5 min (20 μL/min) using a 3–35% acetonitrile gradient in 0.1% formic acid. Collision energy (CE) value for each ionized peptide was determined by Skyline software (ver. 20.2) providing value. MRM-MS data were analyzed by using AB Sciex Analyst software (ver. 1.7.2). Peak picking and determination of peak areas were first performed using DeepMRM (27) and then manually inspected.

All serum specimens were collected with informed patient consent under a protocol approved by the Seoul National University Hospital Institutional Review Board (IRB) Ethics Committee. For verification, serum samples from patients with PDAC, benign pancreatic diseases, and healthy individuals were used. We first identified patients who met the following conditions: 1) those diagnosed with either PDAC or benign pancreatic diseases; 2) those who underwent primary surgical treatment between July 2013 and October 2020; and 3) those having donated their blood samples, obtained one day before surgery, for scientific purposes after providing written informed consent. Meanwhile, patients were excluded if they had any malignancy other than the respective conditions. For the healthy control (HC) group, the samples from healthy individuals who visited the Seoul National University Hospital were obtained.